WO2004004743A1

WO2004004743A1 - Immunostimulation by chemically modified rna

Info

Publication number: WO2004004743A1
Application number: PCT/EP2003/007175
Authority: WO
Inventors: Ingmar Hoerr; Florian VON DER MüLBE; Steve Pascolo
Original assignee: Curevac Gmbh
Priority date: 2002-07-03
Filing date: 2003-07-03
Publication date: 2004-01-15
Also published as: EP2216028A3; EP1806139A2; EP1797886A3; US20050250723A1; EP1685844A3; EP1521585A1; EP1685844A2; CA2490983A1; EP1685844B9; ES2304529T3; DE10229872A1; EP1685844B1; ES2539756T3; US20100303851A1; EP1521585B1; CA2490983C; EP2216028B1; EP2216027A3; DE50309540D1; ATE390926T1

Abstract

The invention relates to an immunostimulating agent containing at least one chemically modified RNA. The invention also relates to a vaccine that contains at least one antigen in conjunction with the immunostimulating agent. The inventive immunostimulating agent and the inventive vaccine are used, in particular, against infectious diseases or cancers.

Description

Immune stimulation through chemically modified RNA

The present invention relates to an immunostimulating agent containing at least one chemically modified RNA. The invention further relates to a vaccine which contains at least one antigen in conjunction with the immune-stimulating agent. The immunostimulating agent according to the invention and the vaccine according to the invention are used in particular against infectious diseases or cancer.

RNA plays a central role in the expression of genetic information in the cell in the form of mRNA, tRNA and rRNA. In addition, however, it was shown in some studies that RNA as such is also involved in the regulation of a number of processes, particularly in the mammalian organism. RNA can take on the role of a communication messenger (Benner, FEBS Lett. 1988, 232: 225-228). Furthermore, an RNA with great homology to an ordinary mRNA was discovered, which, however, is not translated but has a function in the intracellular regulation (Brown et al., Cell 1992, 71: 527-542). Such regulatory RNA is characterized by an incomplete sequence of the ribosome binding site (Kozak sequence: GCCGCCACCAUGG, the AUG forming the start codon (cf. Kozak, Gene Expr. 1991, 1 (2): 117-125), in which it differs from (normal) mRNA In addition, it could be shown that this class of regulatory RNA also occurs in activated cells of the immune system, for example CD4 ⁺ T cells (Liu et al., Genics 1997, 39: 171 -184). In both classic and genetic vaccination, the problem often arises that only a small and therefore inadequate immune response is caused in the organism to be treated or vaccinated. Therefore, so-called adjuvants, ie substances which can increase and / or influence an immune response against an antigen, are often added. Adjuvants that have long been known in the art are, for example, aluminum hydroxide, Freud's adjuvant and others. Such adjuvants, however, produce undesirable side effects, for example very painful irritation and inflammation at the site of application. Toxic side effects, especially tissue necrosis, are also observed. Finally, these known adjuvants only insufficiently stimulate the cellular immune response, since only B cells are activated.

Furthermore, bacterial DNA is known to have an immunostimulating effect due to the presence of unmethylated CG motifs, which is why such CpG-DNA has been proposed as an immune-stimulating agent alone and as an adjuvant for vaccines; see. U.S. Patent 5,663,153. This immunostimulating property of DNA can also be achieved by DNA oligonucleotides that are stabilized by phosphorothioate modification (US Pat. No. 6,239,116). U.S. Patent 6,406,705 finally discloses adjuvant compositions containing a synergistic combination of a CpG oligodeoxyribonucleotide and a non-nucleic acid adjuvant.

However, the use of DNA as an immunostimulating agent or as an adjuvant in vaccines is disadvantageous in several respects. DNA is broken down in the bloodstream only relatively slowly, so that the use of immunosrimulating DNA can lead to the formation of anti-DNA antibodies, which has been confirmed in the mouse animal model (Gilkeson et al., J. Gin. Invest 1995, 95: 1398-1402). The possible persistence of DNA in the organism can thus lead to an overactivation of the immune system, which is known to result in splenomegaly in mice (Montheith et al., Anticancer Drug Res. 1997, 12 (5): 421-432). In addition, DNA can interact with the host genome, in particular by inducing mutations in the host genome. For example, the inserted DNA can be inserted into an intact gene, which is a mutation that represents the function of the endogenous gene hampers or even completely turns off. Such integration events can on the one hand switch off vital enzyme systems for the cell, and on the other hand there is also the risk of the cell thus changed being transformed into a degenerate state if the integration of the foreign DNA changes a gene that is crucial for the regulation of cell growth. Therefore, when using DNA as an immunostimulating agent, a risk of cancer formation cannot be excluded.

Riedl et al. Q. Immunol. 2002, 168 (10): 4951-4959) disclose that RNA bound to an Arg-rich domain of the HBcAg nucleocapsid elicits a Thl -mediated immune response against HbcAg. The Arg-rich domain of the nucleocapsid is similar to protarnins and binds non-specifically nucleic acids.

It is therefore the object of the present invention to provide a new system for improving the stimulation of the limbs in general and the vaccination in particular, which produces a particularly efficient immune response in the patient to be treated or vaccinated, but avoids the disadvantages of known immunostimulants.

This object is achieved by the embodiments of the present invention characterized in the claims.

In particular, an immunostimulating agent is provided according to the invention, containing at least one RNA which has at least one chemical modification. Thus, according to the present invention, the use of the chemically modified RNA for the production of an immunostimulating agent is also disclosed.

The present invention is based on the surprising finding that chemically modified RNA cells of the immune system (primarily antigen-presenting cells, in particular dentritic cells (DC), and also the defense cells, for example in the form of T cells) are particularly strongly activated and so that To stimulate an organism's immune system. In particular, the immuninulatory agent according to the invention, containing the chemically modified RNA, leads to an increased release of immune-regulating agents Cytokines, for example interleukins such as IL-6, IL-12 etc. It is therefore possible, for example, to use the immunostimulating agent of the present invention against infections or cancer diseases, for example by injecting it into the infected organism or the tumor itself. Malignant melanoma, colon carcinoma, lymphoma, sarcoma, small cell lung carcinoma, blastoma, etc. can be mentioned as examples of cancers which can be treated with the immunostimulating agent according to the invention. Furthermore, the immunostimulating agent is advantageous against infectious diseases (e.g. viral infectious diseases such as AIDS (HIV), hepatitis A, B or C, Heφes, Herpes Zoster (Vaή cells), rubella (Rubeola virus), yellow fever, dengue etc.) ( Flavi viruses), flu (influenza viruses), hemorrhagic infectious diseases (Marburg or Ebola viruses), bacterial infectious diseases such as Legionnaires' disease (Legionella), gastric ulcer (Helicobacter), cholera (Vibrio), E. ^ // - infections, Staphylococcal infections, Salmonella infections or streptococcal infections (tetanus), protozoological infectious diseases (malaria, sleeping sickness, leishmaniasis, toxoplasmosis, ie infections by plasmodium, trypanosomes, leishmania and toxoplasmas, or fungal infections, which is neococplasm, by cryptocytoplasm, by Crypt capsulatum, Cocädioides immitis, Blastomyces dermatitidis or Candida albkans) are used).

The term “chemical modification” means that the RΝA contained in the immune stimulant according to the invention is changed by replacing, adding or removing individual or more atoms or groups of atoms in comparison to naturally occurring RΝA species.

The chemical modification is preferably designed such that the R ,A contains at least one analogue of naturally occurring nucleotides.

In a list that is by no means exhaustive, examples of nucleotide analogs which can be used according to the invention are phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguaonsine, 5-methylcytosine and inosine. The preparation of such analogs is known to a person skilled in the art, for example from US Patents 4,373,071, US 4,401,796, US 4,415,732, US 4,458,066, US 4,500,707, US 4,668,777, US 4,973,679, US 5,047,524, US 5,132,418, US 5,153,319, US 5,262,530 and 5,700,642. known. It is particularly preferred if the RNA consists of nucleotide analogs, for example the aforementioned analogs.

Further chemical modifications include, for example, the addition of a so-called “5'-cap” structure, ie a modified guanosine nucleotide, in particular m7G (5 ') ppp (5 ^, (A, G (5') ppp ( 5 ') A and G (5') ρρρ (5 ') G.

According to a further preferred embodiment of the present invention, the chemically modified RNA is a relatively short RNA molecule which, for example, consists of approximately 2 to approximately 1000 nucleotides, preferably approximately 8 to approximately 200 nucleotides, particularly preferably 15 to approximately 31 Nucleotides.

According to the invention, the RNA contained in the immunostimulating agent can be single or double-stranded. Double-stranded RNA with a length of 21 nucleotides in particular can also be used as interference RNA to specifically target genes, e.g. of tumor cells, and thus to specifically kill these cells or to inactivate active genes which are responsible for malignant degeneration (Elbashir et al., Nature 2001, 411, 494-498).

Specific examples of RNA species which can be used according to the invention result if the RNA has one of the following sequences: 5'-UCCAUGACGUUCCUGAUGCU-3 ', 5'-UCCAUGACGUUCCUGACGUU-3' or 5'-UCCAGGACUUCUCUCAGGUU-3 \ It is particularly preferred if the RNA species are phosphorotioate-modified.

If appropriate, the immunostimulating agent according to the invention can contain the chemically modified RNA in conjunction with a pharmaceutically acceptable carrier and / or vehicle.

To further increase the immunogenicity, the immunostimulating agent according to the invention can contain one or more adjuvants. With regard to immune stimulation, a synergistic effect of chemically modified RNA according to the invention and the adjuvant is preferably achieved. "Adjuvans" includes every understand bond that favors an immune response. Depending on the different types of adjuvants, different mechanisms can be considered. For example. form compounds which allow the maturation of the DC, for example upopolysaccharides, TNF or CD40 ligand, a first class of suitable adjuvants. In general, any agent which influences the immune system, of the type of a "danger signal" (LPS, GP96 etc.) or cytokines, such as GM-CFS, can be used as adjuvant, which allow an immune response to be strengthened and / or influenced in a directed manner , If necessary, CpG oligonucleotides can also be used here, although the side effects which may occur, as explained above, must be weighed up. Due to the presence of the immunostimulating agent according to the invention, containing the chemically modified RNA, as the primary immunostimulant, however, only a relatively small amount of CpG-DNA is required (compared to immunostimulation only with CpG-DNA), which is why a synergistic effect of the immunostimulating agent according to the invention and CpG-DNA generally leads to a positive evaluation of this combination. Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins or chemokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-2, INF-, INF-γ, GM-CFS, LT-α or growth factors, e.g. hGH, are preferred. Other known adjuvants are aluminum hydroxide, Freud's adjuvant and the stabilizing cationic peptides or polypeptides mentioned below, such as protamine, and cationic polysaccharides, especially chitosan. Furthermore, lipopeptides such as Pam3Cys are also particularly suitable for use as adjuvants in the immunostimulating agent of the present invention; see. Deres et al, Nature 1989, 342: 561-564.

In addition to being used directly to start an immune reaction, for example against a pathogenic germ or against a tumor, the immunostimulating agent can also advantageously be used to strengthen the immune response against an antigen. The chemically modified RNA can therefore be used to produce a vaccine in which it acts as an adjuvant that favors the specific immune response against the respective antigen or antigens. Another object of the present invention thus relates to a vaccine containing the immunostimulating agent defined above and at least one antigen.

In "classic" vaccination, the vaccine according to the invention or the vaccine to be produced using the chemically modified RNA contains the at least one antigen itself. An "antigen" is to be understood as any structure which involves the formation of antibodies and / or the activation of a cellular one Can elicit an immune response. According to the invention, the terms "antigen" and "immunogen" are therefore used synonymously. Examples of antigens are peptides, polypeptides, including proteins, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, plycolipids and carbohydrates. Antigens include, for example, tumor antigens, viral, bacterial, fungal and protozoological antigens. Surface antigens of tumor cells and surface antigens, in particular secreted forms, are preferred, viral, bacterial, fungal or protozoological pathogens. Of course, the antigen in the vaccine according to the invention can also be in the form of a hapten coupled to a suitable carrier. Suitable carriers are known to a person skilled in the art and include, for example, human serum albumin (HSA), polyethylene glycols (PEG) and others. on. The coupling of the hapten to the carrier is carried out according to methods known in the art, for example in the case of a polypeptide carrier via an amide bond to a Lys residue.

In the case of genetic vaccination using the vaccine according to the invention or the genetic vaccine to be produced using the chemically modified RNA, an immune response is stimulated by introducing the genetic information for the at least one antigen (in this case, therefore, a peptide or polypeptide). Antigen) in the form of a nucleic acid coding for this antigen, in particular a DNA or an RNA (preferably an mRNA), into the organism or into the cell. The nucleic acid contained in the vaccine is translated into the antigen, ie the polypeptide encoded by the nucleic acid or an antigenic peptide is expressed, thereby stimulating an immune response directed against this antigen. When vaccinating against a pathological germ, ie a virus, a bacterium or a protozoological germ, a surface antigen of such a germ is therefore preferred for vaccination with the help of the vaccine according to the invention, containing one for the upper area antigen coding nucleic acid used. In the case of use as a genetic vaccine for the treatment of cancer, the immune response is achieved by introducing the genetic information for tumor antigens, in particular proteins which are only expressed on cancer cells, by administering a vaccine according to the invention which nucleic acid coding for such a cancer antigen contains. This expresses the cancer antigen (s) in the organism, causing an immune response that is effectively directed against the cancer cells.

The vaccine according to the invention is particularly suitable for the treatment of cancer. A tumor-specific surface antigen (TSSA) or a nucleic acid which codes for such an antigen is preferably used. Thus, the vaccine according to the invention can be used for the treatment of the cancers mentioned above with reference to the inventive stimulator. Specific examples of tumor antigens which can be used in the vaccine according to the invention include 707-AP, AFP, ART-4, BAGE, ß-catenin / m, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GplOO, HAGE, HER-2 / new, HLA-A * 0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1, pl90 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, RAGE, RUl or RU2, SAGE, SART-1 or SART-3, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2 and WT1.

Furthermore, the vaccine according to the invention is used against infectious diseases, in particular the infections mentioned above in relation to the immunostimulating agent according to the invention. In the case of infectious diseases, the corresponding surface antigens with the strongest antigenic potential or a nucleic acid coding therefor are preferably used in the vaccine. In the case of the mentioned antigens of pathogenic germs or organisms, in particular in the case of viral antigens, this is typically a secreted form of a surface antigen. Furthermore, according to the invention, preference is given to using polyepitopes or nucleic acids coding therefor, in particular mRNAs, these preferably being polyepitopes of the abovementioned th antigens, in particular surface antigens of pathogenic germs or organisms or tumor cells, preferably secreted protein forms.

In addition, a nucleic acid coding for at least one antigen, which may be contained in the vaccine according to the invention, may also contain, in addition to the section coding for an antigenic peptide or polypeptide, at least one further functional section, for example for a cytokine which promotes the immune response. in particular those coded above from the point of view of the "adjuvant".

As already mentioned, the nucleic acid encoding at least one antigen can be DNA or RNA. In order to introduce the genetic information for an antigen into a cell or an organism, in the case of a DNA vaccine according to the invention, a suitable vector is generally required, which contains a section coding the respective antigen. The vectors of the series pVITRO, pVIVO, pVAC, pBOOST etc. (InvivoGen, San Diego, CA, USA), which are described under the URL http://www.invivogen.com, can be mentioned as specific examples of such vectors. the disclosure content of which is fully incorporated into the present invention.

In connection with DNA vaccines according to the invention, various methods for introducing the DNA into cells, such as, for example, calcium phosphate transfection, polyprene transfection, protoblast fusion, electroporation, microinjection and lipofection, can be mentioned, with lipofection being particularly preferred is.

In the case of a DNA vaccine, however, the use of DNA viruses as a DNA vehicle is preferred. Viruses of this type have the advantage that, due to their infectious properties, a very high transfection rate can be achieved. The viruses used are genetically modified so that no functional infectious particles are formed in the transfected cell.

From the point of view of safety, the use of RNA as nucleic acid coding for at least one antigen is preferred in the vaccine according to the invention. In particular, In particular, RNA does not pose the risk of being stably integrated into the genome of the transfected cell. Furthermore, no vital sequences, such as promoters, are required for effective transcription. In addition, RNA is broken down much more easily in vivo. Probably due to the relatively short half-life of RNA in the bloodstream compared to DNA, no anti-RNA antibodies have so far been detected.

It is therefore preferred according to the invention if the nucleic acid coding for at least one antigen is an mRNA which contains a section coding for the at least one peptide or polypeptide antigen.

However, compared to DNA, RNA in solution is much more unstable. RNA-degrading enzymes, so-called RNAases (ribonucleases), are responsible for the instability. Even the smallest contaminants from ribonucleases are sufficient to completely degrade RNA in solution. Such RNase impurities can generally only be removed by special treatments, in particular with diethyl pyrocarbonate (DEPC). The natural breakdown of mRNA in the cytoplasm of cells is very finely regulated. Several mechanisms are known in this regard. For a functional mRNA, the terminal structure is of crucial importance. The so-called "cap structure" (a modified guanosine nucleotide) is located at the 5 'end and a sequence of up to 200 adenosine nucleotides (the so-called poly A tail) is located at the 3' end. The RNA is recognized as mRNA via these structures and the degradation is regulated. In addition, there are other processes that stabilize or destabilize RNA. Many of these processes are still unknown, but often an interaction between the RNA and proteins seems to be decisive. For example. An "rnRNA surveillance system" was recently described (Hellerin and Parker, Ann. Rev. Genet. 1999, 33: 229 to 260), in which incomplete or nonsense mRNA was recognized by certain feedback protein interactions in the cytosol and the Degradation is made accessible, with a major part of these processes being carried out by exonucleases.

It is therefore preferred to stabilize both the chemically modified RNA according to the invention and the RNA coding for an antigen, in particular an mRNA, which may be present in the vaccine against degradation by RNases. The chemically modified RNA and, if appropriate, the mRNA coding for at least one antigen can be stabilized in that the chemically modified RNA or the possibly present mRNA coding for the antigen with a cationic compound, in particular a polycationic compound, for example a (poly ) cationic peptide or protein, associated or complexed or bound to it. In particular, the use of protamine as a polycationic, nucleic acid-binding protein is particularly effective. Furthermore, the use of other cationic peptides or proteins, such as poly-L-lysine or histones, is also possible. This procedure for stabilizing the modified mRNA is described in EP-A-1083232, the disclosure content of which in this regard is fully included in the present invention. Further preferred cationic substances which can be used to stabilize the chemically modified RNA and / or the mRNA which may be present in the vaccine according to the invention include cationic polysaccharides, for example chitosan. The association or complexation with cationic compounds also improves the transfer of the RNA molecules into the cells to be treated or the organism to be treated.

In the sequences of eukaryotic mRNAs there are destabilizing sequence elements (DSE) to which signal proteins bind and regulate the enzymatic degradation of the mRNA in vivo. Therefore, in order to further stabilize the mRNA contained in the vaccine according to the invention, in particular in the region coding for the at least one antigen, one or more changes are made to the corresponding region of the wild-type mRNA, so that no destabilizing sequence elements are contained. Of course, it is also preferred according to the invention to eliminate any DSE present in the non-translated regions (3'- and / or 5'-UTR) from the mRNA. It is also preferred with regard to the immunostimulating agent according to the invention that the sequence of the chemically modified RNA contained therein does not have any such destabilizing sequences.

Examples of the above DSE are AU-rich sequences ("AURES") which occur in 3'-UTR sections of numerous unstable mRNA (Caput et al., Proc. Natl. Acad. Sei. USA 1986, 83: 1670 to 1674). The RNA molecules contained in the vaccine according to the invention are therefore preferably modified compared to the wild-type mRNA in such a way that they have no such destabilizing sequences. This also applies to those sequence motifs that are recognized by possible endonucleases, for example the sequence GAACAAG, which is contained in the 3 'UTR segment of the gene coding for the transferin receptor (Binder et al, EMBO J. 1994, 13: 1969 until 1980). These sequence motifs are preferably also eliminated from the chemically modified RNA molecules of the immunostimulating agent according to the invention or from the mRNA which may be present in the vaccine according to the invention.

The mRNA molecules which can be contained in the vaccine according to the invention also preferably have a 5'-cap structure. Again, m7G (5 ') ppp (5' (A, G (5 ') ppp (5') A and G (5 ') ppp (5') G can be mentioned as examples of cap structures mRNA, as explained above with regard to the chemically modified RNA, have analogs of naturally occurring nucleotides.

According to a further preferred embodiment of the present invention, the mRNA contains a polyA tail of at least 50 nuclotides, preferably at least 70 nuclotides, more preferably at least 100 nuclotides, particularly preferably at least 200 nuclotides.

For efficient translation of the mRNA coding for at least one antigen, an effective binding of the ribosomes to the ribosome binding site (Kozak sequence: GCCGCCACCAUGG, the AUG forms the start codon) is also required. In this regard, it has been found that an increased A / U content around this site enables a more efficient ribosome binding to the mRNA.

Furthermore, it is possible to insert one or more so-called IRES (internal ribosomal entry side) into the mRNA coding for at least one antigen. An IRES can thus act as the sole ribosome binding site, but it can also be used to provide an mRNA that encodes several peptides or polypeptides that are to be translated independently of one another by the ribosomes (“multicistronic Examples of IRES sequences which can be used according to the invention are those from picornaviruses (for example FMDV), pest viruses (CFFV), polioviruses (PV), encephalo-myocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis -C viruses (HCV), classic swine fever viruses (CSFV), murine leukoma viruses (MLV), Simean immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).

According to a further preferred embodiment of the present invention, the mRNA has stabilization sequences in the 5 'and / or 3' untranslated regions which are capable of increasing the half-life of the mRNA in the cytosol.

These stabilizing sequences can have a 100% sequence homology to naturally occurring sequences which occur in viruses, bacteria and eukaryotes, but can also be partially or completely synthetic in nature. The non-translated sequences (UTR) of the β-globin gene, for example from Homo sapiens or Xenopus laems, can be mentioned as an example of stabilizing sequences which can be used in the present invention. Another example of a stabilization sequence has the general formula (C / U) CCAN _x CCC (U / A) Py _x UC (C / U) CC, which is contained in the 3'UTR of the very stable mRNA required for α-globin , α- (I) collagen, 15-lipoxygenase or encoded for tyrosine hydroxylase (cf. Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Of course, such stabilization sequences can be used individually or in combination with one another or in combination with other stabilization sequences known to a person skilled in the art.

To further increase the translation efficiency of the mRNA which may be present in the vaccine according to the invention, the region coding for the at least one antigen (and also any further coding section which may be present) can have the following modifications compared to a corresponding wild-type mRNA, which are present either alternatively or in combination can.

On the one hand, the G / C content of the region of the modified mRNA coding for the peptide or polypeptide can be greater than the G / C content of the coding region of the for the peptide or polypeptide encoding wild-type mRNA, the encoded amino acid sequence being unchanged from the wild-type.

This modification is based on the fact that the sequence sequence of the region of the mRNA to be translated is essential for an efficient translation of an mRNA. The composition and the sequence of the different nucleotides play an important role. In particular, sequences with increased G (guanosine) / C (cytosine) content are more stable than sequences with increased A (adenosine) / U (uracil) content. Therefore, according to the invention, while maintaining the translated amino acid sequence, the codons compared to the wild-type mRNA are varied in such a way that they contain more G / C nucleotides. Since several codons code for one and the same amino acid (degeneration of the genetic code), the codons most favorable for stability can be determined (alternative codon usage, "codon usage").

Depending on the amino acid to be encoded by the mRNA, different possibilities for modifying the mRNA sequence compared to the wild-type sequence are possible. In the case of amino acids encoded by codons that contain only G or C nucleotides, no modification of the codon is required. The codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) do not require any changes since there is no A or U.

In the following cases, the codons which contain A and / or U nucleotides are changed by substituting other codons which code for the same amino acids but do not contain A and / or U. Examples are: The codons for Pro can be changed from CCU or CCA to CCC or CCG; the argon codons can be from CGU or CGA or AGA or AGG to CGC or

CGG to be changed; the codons for Ala can be changed from GCU or GCA to GCC or GCG; the codons for Gly can be changed from GGU or GGA to GGC or GGG. In other cases, A and U nucleotides cannot be eliminated from the codons, however, it is possible to reduce the A and U content by using codons which contain fewer A and / or U nucleotides. For example:

The codons for Phe can be changed from UUU to UUC; the codons for Leu can be changed from UUA, CUU or CUA to CUC or CUG; the codons for Ser can be changed from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be changed from UAU to UAC; the stop codon UAA can be changed to UAG or UGA; the codon for Cys can be changed from UGU to UGC; the codon His can be changed from CAU to CAC; the codon for gin can be changed from CAA to CAG; the codons for Ile can be changed from AUU or AUA to AUC; the codons for Thr can be changed from ACU or ACA to ACC or ACG; the codon for Asn can be changed from AAU to AAC; the codon for Lys can be changed from AAA to AAG; the codons for Val can be changed from GUU or GUA to GUC or GUG; the codon for Asp can be changed from GAU to GAC; the codon for Glu can be changed from GAA to GAG.

In the case of the codons for Met (AUG) and Trp (UGG), however, there is no possibility of sequence modification.

The substitutions listed above can of course be used individually but also in all possible combinations to increase the G / C content of the modified mRNA compared to the original sequence. For example, all codons for Thr occurring in the original (wild-type) sequence can be changed to ACC (or ACG). However, combinations of the above substitution options are preferably used, for example: Substitution of all codons in the original sequence coding for Thr to ACC (or ACG) and substitution of all codons originally coding for Ser to UCC (or UCG or AGC);

Substitution of all codons coding for Ile to AUC and substitution of all codons originally coding for Lys to AAG and substitution of all codons originally coding for Tyr to UAC;

Substitution of all codons in the original sequence coding for Val to GUC (or GUG) and substitution of all codons originally coding for Glu to GAG and substitution of all codons originally coding for Ala to GCC (or GCG) and substitution of all codons originally coding for Arg to CGC (or CGG);

Substitution of all codons in the original sequence coding for Val to GUC (or GUG) and substitution of all codons originally coding for Glu to GAG and substitution of all codons originally coding for Ala to GCC (or GCG) and substitution of all codons originally coding for Gly to GGC (or GGG) and substitution of all codons originally coding for Asn to AAC;

Substitution of all codons coding for Val in the original sequence to GUC (or GUG) and substitution of all codons originally coding for Phe to UUC and substitution of all codons originally coding for Cys to UGC and substitution of all codons originally coding for Leu to CUG (or CUC) and substitution of all codons originally coding for gin to CAG and substitution of all codons originally coding for pro to CCC (or CCG); etc.

The G / C content of the region coding for the antigenic peptide or polypeptide (or any other further section which may be present) of the mRNA is preferably particularly preferred by at least 7% points, more preferably by at least 15% points increased by at least 20 percentage points compared to the G / C content of the coded region of the wüdtyp mRNA coding for the corresponding peptide or polypeptide. In this context, it is particularly preferred to increase the G / C content of the mRNA modified in this way, in particular in the region coding for the at least one antigenic peptide or polypeptide, as compared to the wild-type sequence.

A further preferred modification of an mRNA which may be identified in the present invention is based on the knowledge that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Therefore, if so-called "rare" codons are increasingly present in an RNA sequence, the corresponding mRNA is translated significantly worse than in the case when codons are present which code for relatively "frequent" tRNAs.

Thus, according to the invention, in the mRNA (which may be contained in the vaccine), the region coding for the antigen (ie the antigen-active peptide or polypeptide) is changed in relation to the corresponding region of the wild-type rnRNA in such a way that at least one codon of the wild-type Sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively common in the cell and carries the same amino acid as the relatively rare tRNA.

This modification modifies the RNA sequences in such a way that codons are inserted for which frequently occurring tRNAs are available.

A person skilled in the art knows which tRNAs occur relatively frequently in the cell and which are relatively rare in comparison; see. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11 (6): 660-666.

By means of this modification, according to the invention, all codons of the wild-type sequence which code for a tRNA which is relatively rare in the cell can each be exchanged for a codon which codes for a tRNA which is relatively common in the cell and which carries the same amino acid as the relative one rare tRNA.

It is particularly preferred to combine the increased, in particular maximum, sequential G / C content in the mRNA as described above with the "frequent" codons. link without changing the amino acid sequence of the antigenic peptide or polypeptide (one or more) encoded by the coding region of the mRNA. This preferred embodiment provides a particularly efficiently translated and stabilized mRNA for the vaccine according to the invention.

The immunostimulating agent according to the invention preferably contains, in addition to the chemically modified RNA, and the vaccine according to the invention, in addition to the immunostimulating agent, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable vehicle. Corresponding ways of appropriately formulating and producing the immunostimulating agent according to the invention and the vaccine are disclosed in "Remington's Pharmaceutical Sciences" (Mack Pub. Co., Easton, PA, 1980), which forms part of the disclosure of the present invention. Suitable carriers for parenteral administration are, for example, sterile water, sterile saline solutions, polyalkylene glycols, hydrogenated naphthalenes and in particular biocompatible lactide polymers, lactide / glycolide copolymer or polyoxyethylene / polyoxypropylene copolymers. Irrine-stimulating agents and vaccines according to the invention can include filling substances or substances such as lactose, mannitol, substances for covalently attaching polymers, such as polyethylene glycol to antigenic haptens according to the invention, peptides or polypeptides, complexing with metal ions or inclusion of materials in or on special preparations of polymer compounds, such as eg polylactate, polyglycolic acid, hydrogel or on liposomes, microemulsion, micelles, unilamellar or multilamellar vesicles, erythrocyte fragments or spheroblasts. The respective embodiments of the stimulation agent and the vaccine are selected depending on the physical behavior, for example with regard to solubility, stability, bioavailability or degradability. Controlled or constant release of the active ingredient components according to the invention in the vaccine or in the immunostimulating agent includes formulations based on lipophilic depots (eg fatty acids, waxes or oils). Within the scope of the present invention, coatings of immunostimulatory substances and vaccine substances or compositions according to the invention, containing such substances, namely coatings with polymers (for example polyoxamers or polyoxamines) are also disclosed. Furthermore, immunostimulating or vaccine substances or compositions according to the invention can have protective effects. Have layers, such as protease inhibitors or permeability enhancers. Preferred carriers are typically aqueous carrier materials using water for injection (WFI) or water buffered with phosphate, citrate, HEPES or acetate, etc., and the pH typically to 5.0 to 8.0, preferably 6.5 to 7 , 5, is set. The carrier or vehicle will preferably also contain salt components, for example sodium chloride, potassium chloride or other components which make the solution, for example, isotonic. In addition to the constituents mentioned above, the carrier or the vehicle can also contain additional components, such as human serum alburnine (HSA), polysorbate 80, sugar or amino acids.

The manner of administration and the dosage of the immunostimulating agent according to the invention and the vaccine according to the invention depend on the type of disease to be controlled, possibly its stage, the antigen (in the case of the vaccine) as well as the body weight, age and gender of the patient from.

The concentration of the chemically modified RNA as well as of the coding nucleic acid optionally contained in the vaccine in such formulations can therefore vary within a wide range from 1 μg to 100 mg / ml. The immunostimulating agent according to the invention and the vaccine according to the invention are preferably administered to the patient parenterally, for example intravenously, intraarterially, subcutaneously, intramuscularly. It is also possible to administer the immunostimulating agent or the vaccine topically or orally.

Thus, according to the invention, a method for the prevention and / or treatment of the abovementioned diseases is also provided, which comprises administering the immunostimulating agent according to the invention or the vaccine according to the invention to a patient, in particular to a person.

The figures show:

1 shows results for the stimulation of the maturation of dendritic cells (DC) of the mouse by chemically modified RNA according to the invention in comparison with mRNA Protamine-associated mRNA and DNA. DC of the mouse were analyzed with 10 μg / ml mRNA (pp65 for pp65-mRNA, ß-Gal for ß-galactosidase-mRNA), protamine-stabilized mRNA (Protamin + pp65, Protamin + ß-Gal), DNA (CpG DNA 1668, DNA 1982 and CpG DNA 1826) as well as phosphoshioate-modified RNA (RNA 1668, RNA 1982 and RNA 1826) and stimulated the DC activation by measuring the release of IL-12 (FIG. 1A) and IL-6 (FIG. 1B) Cytokine ELISA determined. In both test series, medium without nucleic acid samples and medium with added protamine served as negative controls. Lipopolysaccharide (LPS) was used as a positive comparison. The oligodeoxyrebonucleotides (ODN) CpG DNA 1668 and CpG DNA 1826 each contain a CpG motif. Such ODNs are known to stimulate DC (see U.S. Patent 5,663,153). The ODN DNA 1982 has the same sequence as the CpG DNA 1826, except that the CpG motif was removed by an exchange from C to G. The sequence of the oligoribonucleotides CpG RNA 1668, RNA 1982 and CpG RNA 1826 stabilized by phosphorothioate modification corresponds to the above-mentioned comparison ODN of the respective identification number. In comparison to normal mRNA, the protamine-stabilized mRNA species show only a weak activation of the DC. A much stronger interleukin release, however, is caused in both experiments by the phosphorothioate-modified oligoribonucleotides according to the invention, the values of which are comparable to those of the positive control (LPS). In comparison to protamine-associated mRNA, the stimulation by phosphorothioate-modified oligoribonucleotides results in a more than doubled release of IL-12 or IL-6. This surprisingly strong interleukin release due to the oligoribonucleotides according to the invention is also independent of CpG motifs, as the comparison of the phosphorothioate-modified oligoribonucleotide RNA 1982 according to the corresponding ODN DNA 1982 shows. The ODN DNA 1982 does not produce any interleukin release in the DC, while RNA 1982 causes an interleukin release which is comparable to the positive control LPS in the case of IL-12 and even exceeds it in the case of LL-6.

FIG. 2 shows the results of the determination of the expression of a surface activation marker (CD86) in DC, which were loaded with the samples as previously described in FIG. 1. were acted. To determine CD86 expression, part of the DC was labeled with an anti-CD86-specific monoclonal antibody 3 days after treatment of the DC with the specified samples and the percentage of cells expressing CD86 was determined by flow cytometry. Significant CD86 expression is only observed in the comparison ODN which have a CpG motif and the phosphorothioate-modified RNA species according to the invention. However, all values of the nucleic acid stimulants were clearly below the positive control (LPS). Furthermore, the CD86 determination confirms that, in contrast to DNA species, the DC activation caused by the phosphorothioate-modified RNA according to the invention is independent of CpG motifs: while the CpG-free ODN DNA 1982 does not cause CD86 expression, the corresponding is Phosporthioat-modified oligoribonucleotide RNA 1982 a CD86 expression detected in 5% of the DC.

FIG. 3 shows the results of an AU reaction test using DC, which were activated in vitro with the samples indicated on the x-axis (cf. also FIG. 1). Three days after stimulation, the DC were added to fresh spleen cells from an allogeneic animal and used six days later in a cytotoxicity test in which the release of ⁵¹ Cr was measured in target cells (P 815) compared to control cells (EL 4) , The target or control cells were plated in a constant amount and then incubated for 4 hours with three different dilutions of the spleen cells (effector cells) co-cultivated with the DC, so that there is a ratio of effector cells (E) to target cells (or control cells) ) (T) of 41: 1, 9: 1 and 2: 1, respectively. On the y-axis, the specific kill is given in percent, which is calculated as follows: [(measured radioactivity released - spontaneously released radioactivity) / (maximum release of radioactivity - spontaneously released radioactivity)] x 100. ß-Ga associated with protamine ktosidase-mRNA stimulated DC can only cause 20% specific target cell kill by the effector cells at the lowest dilution. In contrast, DC stimulated by phosphorothioate-modified oligoribonucleotides cause almost 60%, ie approximately tripled, specific killing of the target cells by the effector cells at the lowest dilution. This value is comparable to that of the positive control (LPS) and one containing a CpG motif Comparative ODN (CpG DNA 1668). In contrast, an ODN without a CpG motif (DNA 1982) is inactive, which confirms the results from the previous experiments according to FIG. 1 and FIG. 2. pp65-mRNA (without protamine), ß-galactosidase mRNA (without protamine) as well as protamine and medium alone do not cause specific killings.

FIG. 4 shows results for the stimulation of the maturation of dendritic cells (DC) of B6 mice in comparison to MyD88 knock-out mice by chemically modified oligoribonucleotides according to the invention and comparison ODN. The stimulation with medium only served as a negative control. The stimulation was carried out as previously described in FIG. 1, and the DC activation was determined by measuring the release of IL-12 (FIG. 4A) and IL-6 (FIG. 4B) by means of a cytokine ELISA. In FIG. 4A the IL-12 concentration in ng / ml is plotted on the y-axis, while in FIG. 4B the absorption at 405 nm (absorption maximum of the detection reagent) is plotted on the y-axis, which is the interleukin Concentration is directly proportional. The protein MyD88, a protein from the signaling cascade of the so-called "toll-like receptors" (TLR), is switched off in MyD88 mice. For example, TLR-9 is known to mediate the activation of DC by CpG DNA. DCs from B6 wild-type mice are activated by the phosphorothioate-modified oligoribonucleotides CpG RNA 1688 and RNA 1982 and, as expected, by the comparison ODN CpG DNA 1668. The ODN DNA 1982 (without CpG motif) is again ineffective. In contrast, none of the samples can cause significant release of LL-12 or 11-6 at DC from MyD88 mice. MyD88 therefore appears to be required for the activation of DC by the chemically modified oligoribonucleotides according to the invention and by CpG-ODN.

5 shows results of the stimulation of DC by the chemically modified oligoribonucleotide RNA 1982 according to the invention and two comparison ODNs which were incubated for 2, 26 or 72 h at 37 ° C. with medium which was not RNase before being used for DC activation - was free. For comparison, a sample was used without previous incubation (t = 0). The samples marked "1: 1" were diluted 1: 1 with buffer compared to the other samples. DC activation was again measured by determining the release of LL-12 (FIG. 5A) and IL-6 (FIG. 5B) using a cytokine ELISA. DC activation by CpG-DNA is from a previous incubation tion with medium independent. As expected, the comparison ODN without CpG motif does not lead to any interleukin release. With the oligoribonucleotide RNA 1982 according to the invention, a clear release of interleukin is measured without incubation with medium (t = 0). Already after 2 h of incubation at 37 ° C. with non-RNase-free medium, no significant interleukin release is observed in the stimulation test with the oligoribonucleotide according to the invention.

FIG. 6 shows the result of a similar experiment as shown in FIG. 5B, but a more precise time course of the effect of RNA degradation on DC stimulation was recorded: The chemically modified oligoribonucleotide RNA 1982 was again used to stimulate DC is used and the activation of the DC is determined by measuring the IL-6 release. Before stimulation, the oligoribonucleotide was incubated with non-RNase-free medium for 15, 30, 45 or 60 minutes as described above in FIG. 5. A sample that was not incubated with the medium (t = 0) was again used for comparison. ODN CpG DNA 1668 was used as positive control and medium alone as negative control. Without prior incubation with non-RNase-free medium, there is again strong DC activation by the chemically modified RNA according to the invention, as evidenced by the IL6 concentration of over 5 ng / ml. This value drops to slightly over 2 ng / ml within an hour of incubation under RNA degradation conditions. This shows that the chemically modified RNA is degraded much faster than DNA species under physiological conditions, but the half-life is obviously sufficiently long for the immunostimulating effect according to the invention to be developed.

7 shows results for the stimulation of the proliferation of mouse B cells with phosphorothioate-modified ribonucleotides according to the invention (CpG RNA 1668, CpG RNA 1826 and RNA 1982) in comparison to DNA species (with CpG motif: CpG DNA 1668 and CpG DNA 1826; without CpG motif: DNA 1982). Medium alone without a nucleic acid sample served as a control. ODN with CpG motif lead to a very strong B cell proliferation with almost 90% proliferating B cells. The ODN DNA 1982 (without CpG motif), which has proven to be inactive with regard to DC stimulation (cf. FIGS. 1 to 5), also called moderate B cell proliferation (almost 20% proliferating Cells). In contrast, the stimulation of the B cells by the chemically modified oligoribonucleotides according to the invention led to a percentage of proliferating B cells which is in the range or even below the negative control (in any case <10% proliferating cells).

8 shows results of an investigation in vivo on the effect of chemically modified RNA according to the invention in comparison with DNA on the spleen of mice. The respective nucleic acid species were injected subcutaneously with an antigen mixture (peptide TPHARGL ("TPH") + ß-galactosidase ("ß-Gal"). After 10 days, the spleens of the mice were removed and weighed. On the y-axis the spleen weight is plotted in g The bars each show the mean of two independent experiments, while the spleen weight in the mice treated with the chemically modified RNA + antigen mixture according to the invention is unchanged from the control (PBS) at approximately 0.08 g , mice injected with DNA + antigen mixture show pronounced splenomegaly, which is shown by an average spleen weight of over 0.1 g.

The following examples illustrate the present invention without restricting it.

Examples

The following materials and methods were used to carry out the examples below:

1. Cell culture

Dendritic cells (DC) were obtained by rinsing the hind bone marrow from BLAB / c, B6 or MyD88 knock-out mice with medium, treatment with Gey's solution (for lysis of the red blood cells) and filtration through a cell sieve. The cells were then in IMDM for 6 days containing 10% heat inactivated fetal calf serum (FCS; from PAN), 2 mM L-glutamine (from Bio Whittaker), 10 mg / ml streptomycin, 10 U / ml penicillin (PEN-STREP, from Bio Whittaker) and 51 U / ml GM-CFS (hereinafter referred to as "complete medium"), cultured in 24-well culture plates. After two and four days, respectively, the medium was removed and an equivalent volume of fresh medium containing the above-mentioned concentration of GM-CFS was added.

2. Activation of the DC

After 6 days, the DC were transferred to a 96-well culture plate, with 200,000 cells in 200 μl of complete medium added to each well. The nucleic acid samples (DNA, chemically modified RNA, mRNA or protamine-stabilized RNA) were added at a concentration of 10 μg / ml.

3, RNA degradation conditions

5 .mu.l of the corresponding nucleic acid samples (2 .mu.g / .mu.l DNA, unmodified RNA or RNA chemically modified according to the invention) were in 500 .mu.l complete medium and for 2, 26 or 72 h or 15, 30, 45 or 60 min at 37 ° C incubated. A non-incubated sample (t = 0) served as a control. DC were then stimulated with the samples as described under point 2 above.

4. Cytokine ELISA

17 hours after the addition of the respective stimulant, 100 μl of the supernatant were removed and 100 μl of fresh medium were added. ELISA plates (Nunc Maxisorb) were coated overnight with capture antibodies (Pharmingen) in binding buffer (0.02% NaN ₃ , 15 mM Na ₂ CO ₃ , 15 mM NaHCO ₃ , pH 9.7). Non-specific binding sites were saturated with phosphate-buffered saline (PBS) containing 1% serum albumin (BSA). Thereafter, 100 μl of the respective cell culture supernatant were added to each well treated in this way and incubated at 37 ° C. for 4 hours. Biotinylated antibody was added after 4 washes with PBS containing 0.05% Tween-20. The Detection reaction was started by adding streptavidin-coupled radish peroxidase (HRP-streptavidin) and the substrate ABTS (measurement of the absorption at 405 nm).

5. Flow cytometry

For single-color flow cytometry, 2 × 10 ⁵ cells were incubated for 20 minutes at 4 ° C. in PBS, containing 10% FCS, with FITC-conjugated, monoclonal anti-CD86 antibody (Beceton Dickinson) at a suitable concentration. After washing twice and fixing in 1% formaldehyde, the cells were analyzed with a FACScalibur flow cytometer (Becton Dickinson) and the CellQuestPro software.

6; AU reaction test by ⁵¹ Cr release

B6 mice (C57bl6, H-2 ^d haplotype) were incubated with the DC of BLAB / c mice (H-2 ^d haplotype) stimulated according to item 2 above in a ratio of 1: 3 for 5 days and used as effector tools.

5,000 EL-4 cells (as a control) and P815 cells (as a target) were cultivated in 96-well plates in IMDM with 10% FCS and loaded with ⁵¹ Cr for one hour. The ⁵¹ Cr-labeled cells were incubated with the effector cells for 5 hours (final volume 200 μl). There were 3 different ratios of effector and Control lines to target lines (E / T) examined: E / T = 41, 9 or 2. To determine the specific kill, 50 μl of the supernatant were removed and the radioactivity using a solid phase scintillation plate (Luma Plate-96, Packard) and a scintillation counter for microtiter plates (1450 Microbeta Plus). The percentage of ⁵¹ Cr release was determined based on the amount of ⁵¹ Cr (A) released into the medium and with the spontaneous ⁵¹ Cr release of target cells (B) and the total ⁵¹ Cr content of target cells (C) were lysed with 1% Triton-X-100, and the specific killing results from the following formula:% killing = [(A - B) / (C - B)] x 100. 7. B-ZeU trial test

Fresh spleens from a mouse were washed twice with 10 ml of PBS and taken up in a concentration of 1 × 10 ⁷ cells / ml in PBS. CSFE (FITC-labeled) was added to a final concentration of 500 nM and incubated for 3 minutes. It was then washed twice with medium. One undyed and one colored sample was examined in a flow cytometer (FACScalibur; Becton Dickinson). CpG DNA or RNA in a concentration of 10 μg / ml was added to 200,000 cells / well of a culture plate with 96 wells (U-shaped bottom) in 200 μl medium. On day 4 after stimulation, the cells were stained with B220 CyChrome and CD 69 PE and analyzed in the FACS.

; In iWO study on SplenomegaUe

50 μg of chemically modified RNA or reference ODN were injected subcutaneously with an antigen mixture (100 μg peptide TPHARGL + 100 μg β-galactosidase) in each 200 μl PBS in BALB / c mice (two mice were used for each sample). After 10 days, the spleens were removed from the mice and weighed.

9. Sequences of the nucleic acids used

OUgodeoxyribonucleotides (ODN):

CpG DNA 1668: 5'-TCCATGACGTTCCTGATGCT-3 'CpG DNA 1826: 5'-TCCATGACGTTCCTGACGTT-3'

DNA 1982: 5'-TCCAGGACTTCTCTCAGGTT-3 '

OHgoribonucleotides (modified phosporthioate):

CpG RNA 1668: 5'-UCCAUGACGUUCCUGAUGCU-3 '

CpG RNA 1826: 5'-UCCAUGACGUUCCUGACGUU-3 '

RNA 1982: 5'-UCCAGGACUUCUCUCAGGUU-3 ' example 1

In order to determine the ability of various nucleic acid species to stimulate the maturation of DC, DC were obtained from BALB / c mice and treated with the OHgonucleotides given under point 6 above. Protamine-stabilized β-galactosidase mRNA and pp65-RNA were used as further samples. The release of LL-12 or IL-6 by the stimulated DC was determined by means of ELISA. The stimulation of DC by means of protamine-associated mRNA resulted in a weak interleukin release. In contrast, the interleukin release caused by the phosphorothioate-modified RNA species according to the invention was significantly greater and was even comparable to the positive control (stimulation by LPS) (FIGS. 1A and 1B). The comparative ODN, which contained a CpG motif, showed an expected interleukin release by the DC, but the interleukin release was significantly lower compared to the value caused by the sequence corresponding to the inventive RNA species (FIG. 1A and 1B).

In order to confirm the induction of the maturation of the DC demonstrated by means of cytokine ELISA, the expression of a surface marker specific for mature DC (CD86) was determined by means of flow cytometry. Phosphorothioate-modified RNA species according to the invention, but not mRNA or protamine-associated mRNA, were able to produce clear CD86 expression (FIG. 2).

Example 2

Furthermore, it was investigated whether the DC activated by the chemically modified, immunostimulating RNA species are capable of eliciting an immune response in an autogenous system (FIG. 3). For this purpose, mouse spleen cells (B6) were activated with the stimulated DC and brought together as effector cells with external target cells (P815), the killing of the target cells being determined with the aid of a Cr release test. Three different dilutions of effector lines were brought into contact with a constant number of target lines. In comparison to protamine-stabilized mRNA, phosphorothioate-modified RNA is then much more capable of causing the maturation of DC to activated cells that can start an immune response through effector cells. Surprisingly, it can be found that DC activated by phosphorothioate RNA can trigger an immune response that is as strong as that triggered by ODN that have CpG motifs.

Example 3

It is known that the activation of DC by CpG-DN is mediated via TLR-9 ("toU-like receptor 9") (Kaisho et al., Trends Immunol. 2001, 22 (2): 78-83) , It was therefore investigated whether the TLR signal cascade is also involved in the DC activation brought about by the chemically modified, immunostimulant RNA. For this purpose, the activation of DC from B6 female-type mice was compared with that of DC from B6 mice lacking the protein MyD88 on the basis of the release of LL-12 and IL-6. MyD88 is involved in the TLR-9 signal cascade. The strong release of IL-12 and IL-6 by DC from the B6-WUdtyp mice confirmed the results of Example 1 (see FIGS. 4A and B, black bars). In contrast, stimulation of DC from MyD88 knock-out mice with the same samples led to no activation (cf. FIGS. 4A and B, white bars). These results show that MyD88, and thus the TLR-9 signaling cascade, is required for both CpG-DNA-mediated and chemically modified RNA-mediated DC activation.

Example 4

In order to investigate whether chemically modified RNA according to the invention is subject to rapid degradation and therefore does not pose a risk of persistence in the organism, OUgoribonucleotides according to the invention were used under RNA degradation conditions (37 ° C., untreated medium, ie not RNase-free) for 2 Incubated for 26, 72 or 72 h and only then subjected to the stimulation test with DC. After a two-hour incubation under RNA degradation conditions, the According to the chemically modified RNA according to the invention, activation of the DC was no longer observed, as is shown by the lack of LL-12 (FIG. 5A) and IL-ό release (FIG. 5B). In contrast, the previous incubation of CpG DNA species has no influence on their effectiveness in DC activation. This shows that the chemically modified RNA according to the invention is broken down after a relatively short time, which avoids persistence in the organism that can occur with DNA.

The chemically modified RNA according to the invention, however, is not degraded so quickly that it can no longer develop its innate effects. To demonstrate this, the above experiment was repeated with a phosphorothioate-modified Ogoribonucleotide (RNA 1982) according to the invention, but the incubation under RNA degradation conditions was only carried out for 15, 30, 45 and 60 min. As the release of LL-6 by the DC stimulated afterwards shows, there is also a clear DC activation after an hour of incubation under RNA degradation conditions (FIG. 6).

Example 5

The induction of a splenomegaHe, which is essentially due to a strong activation of the B-ZeU-ProUferation, constitutes a major obstacle to the use of CpG-DNA as an immunostimulating adjuvant in vaccines (see Monteith et al., See above). It was therefore checked by means of a B-ZeU ProUferation test whether the chemically modified RNA according to the invention has an effect on the B-ZeU ProUferation. In the B-ZeU ProUferation test, an expected high number of ProUferieren ZeUen in the FaU of stimulation with CpG-DNA was detected. In contrast, surprisingly, chemically modified RNA according to the invention (regardless of any CpG motifs present in the sequence) was completely ineffective in this regard (FIG. 7).

In order to confirm this surprisingly positive property of the chemically modified RNA according to the invention in vivo, a test vaccine containing a phosphorothioate ogoribonucleotide (RNA 1982) according to the invention and an antigen mixture of a peptide and β-galactosidase was prepared and injected subcutaneously into mice. Served as a comparison a corresponding DNA test vaccine, which contained the same antigen mixture in conjunction with a CpG ODN (CpG DNA 1826). After 10 days, the spleens were removed from the mice and weighed. Compared to the negative control (PBS), there was a significant increase in spleen weight in mice treated with the DNA test vaccine. In contrast, no splenomegaUe was determined in mice treated with the RNA test vaccine according to the invention, since the spleen weight was unchanged compared to the negative control (FIG. 8). These results show that when the chemically modified RNA is used according to the invention as an immunostimulating agent or as an adjuvant in vaccines, there are no side effects associated with an undesirable B-ZeU-ProUferation.

In summary, it can be said that chemically modified RNA causes the maturation of DC in vitro. The examples above show that chemically modified RNA, here in the form of short (e.g. 20-mer) synthetic OUgoribonucleotides (which are phosphorothioate-modified), activates immature DC and thus causes their maturation, as determined by determining the specific cytokine release ( Fig. 1) and the expression of surface activation markers (Fig. 2) is shown. The DC activation caused by the chemically modified RNA is significantly stronger than that caused by a mixture of mRNA and the polycationic compound protamine, which is known to be associated with the RNA and thus protect it from nucleases. The DCs matured by stimulation with chemically modified RNA according to the invention can start an immune response by effector cells, as demonstrated by a Cr ⁵¹ release test in an external system (FIG. 3). DC activation by the chemically modified RNA according to the invention is presumably carried out via a TLR-mediated signal cascade (FIG. 4).

Bacterial DNA is known to have an immunostimulatory effect due to the presence of non-methylated CG motifs; see. U.S. Patent 5,663,153. This property of DNA can be simulated with DNA OUgonucleotides stabilized by phosphorionate modification (US Pat. No. 6,239,116). RNA, which is complexed by positively charged proteins, is known to have an immune-stimulating effect (Riedl et al., 2002, see above). The present invention could shows that RNA, which is chemically modified, is a much more effective immune-stimulating agent compared to other, for example protamine-complexed, RNA. In contrast to DNA, no CpG motifs are required with such chemically modified RNA OHgonucleotides. In contrast to the 20-mer ribonucleotides, free phosphorothioate nucleotides (not shown) are not immunostimulatory.

However, the chemically modified immunostimulatory RNA of the present invention is superior in particular to the intuition-destroying DNA in that RNA is broken down more quickly and thus removed from the patient's body, which is why the risk of persistence and causing serious side effects is reduced or avoided ( 5 and 6). For example, the use of immunostimulating DNA as an adjuvant for vaccines can induce the build-up of anti-DNA antibodies, the DNA can persist in the organism, which can lead, for example, to an overactivation of the immune system, which is known to result in a splenomegaUe in mice (Montheith et al., 1997, see above). The splenomegaUe caused by DNA adjuvants is essentially due to a stimulation of the B-ZeU proUferation, which does not occur with RNA adjuvants according to the invention (FIGS. 7 and 8). In addition, DNA can interact with the host genome, in particular by inducing mutations by integration into the host genome. AU these high risks can be avoided when using chemically modified RNA for the production of immune-stimulating agents or vaccines, in particular for vaccination against or for the treatment of cancer or infectious diseases, with better or comparable immune stimulation.

Claims

claims

1. Immune-stimulating agent containing at least one RNA which has at least one chemical modification.

2. Agent according to claim 1, characterized in that at least one nucleotide of the RNA is an analog of naturally occurring nucleotides.

3. Composition according to claim 2, characterized in that the RNA consists of nucleotide analogs.

4. Composition according to claim 2 or 3, characterized in that the analog is selected from the group consisting of phosphorothioates, phosphoramidates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.

5. Composition according to claim 4, characterized in that the analog is a phosphorothioate.

6. Agent according to one of claims 1 to 5, characterized in that the RNA consists of 2 to about 1000 nucleotides.

7. Composition according to claim 6, characterized in that the RNA consists of about 8 to about 200 nucleotides.

8. Composition according to claim 7, characterized in that the RNA consists of about 15 to about 31 nucleotides.

9. Composition according to one of claims 1 to 8, characterized in that the

RNA the sequence 5'-UCCAUGACGUUCCUGAUGCU-3 \ 5'-UCCAGGACUUCUCUCAGGUU-3 'or 5 CCAUGACGUUCCUGACGUU-3'.

10. Composition according to one of claims 1 to 9, characterized in that it contains at least one adjuvant.

11. Composition according to claim 10, characterized in that the adjuvant from the group consisting of cytokines, lipopeptides and CpG-OUgonucleotiden.

12. Composition according to one of claims 1 to 11, further comprising a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable vehicle.

13. Composition according to one of claims 1 to 12, for the prevention and / or treatment of infectious diseases or cancer.

14. Vaccine containing the immunostimulating agent according to one of claims 1 to 12 and at least one antigen.

15. Vaccine according to claim 14, characterized in that the antigen from the

Group consisting of peptides, polypeptides, ZeUen, ZeUextracts, polysaccharides, polysaccharide conjugates, lipids, GlykoUpiden and carbohydrates, is selected.

16. Vaccine according to claim 15, characterized in that the peptide or polypeptide antigen in the form of a nucleic acid coding therefor.

17. Vaccine according to claim 16, characterized in that the nucleic acid is an mRNA.

18. Vaccine according to claim 17, characterized in that the mRNA is stabilized and / or optimized for translation.

19. Vaccine according to one of claims 14 to 18, characterized in that the antigen is selected from tumor antigens and antigens from viruses, bacteria, fungi and protozoa.

20. Vaccine according to claim 19, characterized in that the viral, bacterial, fungal or protozoological antigen comes from a secreted protein.

21. Vaccine according to claim 19 or 20, characterized in that the antigen is a polyepitope of tumor antigens or antigen of viruses, bacteria, fungi or protozoa.

22. Vaccine according to one of claims 14 to 21 for vaccination against infectious diseases or cancer.

23. Use of an RNA as defined in any one of claims 1 to 10 for the manufacture of an immunostimulating agent for the prevention and / or treatment of infectious diseases or cancer.

24. Use of an RNA as defined in any one of claims 1 to 10 for

Manufacture of a vaccine for vaccination against infectious diseases or cancer.